In recent years, the interest in manufacturing and commercializing complex oligosaccharides including secreted oligosaccharides has increased significantly due to the roles of these compounds in numerous biological processes in living organisms. Secreted oligosaccharides such as human milk oligosaccharides (HMOs), mucin oligosaccharides and Lewis type oligosaccharides have become important potential products for nutrition and therapeutic uses. As a result, low cost ways of producing industrially these oligosaccharides, particularly HMOs, have been sought.
To date, the structures of at least 115 HMOs have been determined (see Urashima et al.: Milk Oligosaccharides, Nova Biomedical Books, New York, 2011, ISBN: 978-1-61122-831-1), and considerably more are probably present in human milk. The thirteen core structures identified to date, for the 115 HMOs, are listed in Table 1.
TABLE 1Core HMO structuresNoCore nameCore structure1lactose (Lac)Galβ1-4Glc2lacto-N-tetraoseGalβ1-3GlcNAcβ1-3Galβ1-4Glc(LNT)3lacto-N-neotetraoseGalβ1-4GlcNAcβ1-3Galβ1-4Glc(LNnT)4lacto-N-hexaoseGalβ1-3GlcNAcβ1-3(Galβ1-(LNH)4GlcNAcβ1-6)Galβ1-4Glc5lacto-N-neohexaoseGalβ1-4GlcNAcβ1-3(Galβ1-(LNnH)4GlcNAcβ1-6)Galβ1-4Glc6para-lacto-N-hexaoseGalβ1-3GlcNAcβ1-3Galβ1-(para-LNH)4GlcNAcβ1-3Galβ1-4Glc7para-lacto-N-Galβ1-4GlcNAcβ1-3Galβ1-neohexaose4GlcNAcβ1-3Galβ1-4Glc(para-LNnH)8lacto-N-octaoseGalβ1-3GlcNAcβ1-3(Galβ1-(LNO)4GlcNAcβ1-3Galβ1-4GlcNAcβ1-6)Galβ1-4Glc9lacto-N-neooctaoseGalβ1-4GlcNAcβ1-3(Galβ1-(LNnO)3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6)Galβ1-4Glc10iso-lacto-N-octaoseGalβ1-3GlcNAcβ1-3(Galβ1-(iso-LNO)3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6)Galβ1-4Glc11para-lacto-N-octaoseGalβ1-3GlcNAcβ1-3Galβ1-(para-LNO)4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc12lacto-N-neodecaoseGalβ1-3GlcNAcβ1-3[Galβ1-(LNnD)4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAcβ1-6]Galβ1-4Glc13lacto-N-decaoseGalβl-3GlcNAcβ1-3[Galβ1-(LND)3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAcβ1-6]Galβ1-4Glc
A few HMOs have recently been synthesized, for example, by hydrogenating their benzyl glycoside precursors after removing other protecting groups from such precursors and then isolating (e.g. by crystallization) the HMOs (WO 2011/100979, WO 2011/100980, WO 2012/007585, WO 2012/007588, WO 2012/113405, WO 2012/127410, WO 2012/155916, WO 2012/156897, WO 2012/156898, WO 2013/044928, WO 2013/091660).
Direct fermentative production of HMOs, especially of short chain trisaccharides, has recently become practical (see Han et al. Biotechnol. Adv. 30, 1268 (2012) and references cited therein). Such fermentation technology has used a recombinant E. coli system wherein one or more types of glycosyl transferases originating from viruses or bacteria have been co-expressed to glycosylate exogenously added lactose, which has been internalized by the LacY permease of the E. coli. However, the use of more than one type of glycosyl transferase for the production of oligosaccharides of four or more monosaccharide units, like LNnT or fucosylated LNnT, has always led to the formation of a complex mixture of oligosaccharides. This is believed to have been due to the overglycosylation of the diverse intermediates produced from the lactose feed as a result of different relative activities of the different glycosyl transferases (WO 01/04341, Dumon et al. Glycoconj. J. 18, 465 (2001), Priem et al. Glycobiology 12, 235 (2002), Dumon et al. Biotechnol. Prog. 20, 412 (2004), M. Randriantsoa: Synthèse microbiologique des antigènes glucidiques des groupes sanguins, Thèse de Doctorat soutenue le Sep. 30, 2008 a I'Université Joseph Fourier, Grenoble, France).
An alternative fermentation method has involved the use of a recombinant E. coli expressing a β-1,3-GalNAc transferase for glycosylating a trisaccharide, globotriose to make globotetraose or globopentaose (Antoine et al. Biochimie 87, 197 (2005), Randriantsoa et al. FEBS Letters 581, 2652 (2007)).
However, there has been a continuing need for an efficient method of making fucosylated, sialylated and N-acetyl-glucosaminylated oligosaccharides, particularly HMOs, of four or more monosaccharide units.